Influence of Animal Heating on PET Imaging Quantification and Kinetics: Biodistribution of 18F-Tetrafluoroborate and 18F-FDG in Mice.

Different environmental conditions under anesthesia may lead to unstable homeostatic conditions in rodents and therefore may alter kinetics. In this study, the impact of different heating conditions on PET imaging quantification was evaluated. Methods: Two groups of 6 adult female BALB/c nude mice with subcutaneously implanted tumors underwent microPET imaging after injection of 18F-labeled tetrafluoroborate or 18F-FDG. Dynamic scans were acquired under optimal and suboptimal heating conditions. Time-activity curves were analyzed to calculate uptake and washout time constants. Results: With 18F-labeled tetrafluoroborate, optimal animal heating led to a stable heart rate during acquisition (515 ± 35 [mean ± SD] beats/min), whereas suboptimal heating led to a lower heart rate and a higher SD (470 ± 84 beats/min). Both uptake and washout time constants were faster (P < 0.01) in animals maintained with optimal heating. Conclusion: Although the difference in heart rates was slight, optimal heating yielded significantly faster uptake and washout kinetics than suboptimal heating in all organs for both tracers.

CXCR4 antagonists suppress small cell lung cancer progression.

Small cell lung cancer (SCLC) is an aggressive tumor with poor prognosis due to early metastatic spread and development of chemoresistance. Playing a key role in tumor-stroma interactions the CXCL12-CXCR4 axis may be involved in both processes and thus represent a promising therapeutic target in SCLC treatment. In this study we investigated the effect of CXCR4 inhibition on metastasis formation and chemoresistance using an orthotopic xenograft mouse model. This model demonstrates regional spread and spontaneous distant metastases closely reflecting the clinical situation in extensive SCLC. Tumor engraftment, growth, metabolism, and metastatic spread were monitored using different imaging techniques: Magnetic Resonance Imaging (MRI), Bioluminescence Imaging (BLI) and Positron Emission Tomography (PET). Treatment of mice bearing chemoresistant primary tumors with the specific CXCR4 inhibitor AMD3100 reduced the growth of the primary tumor by 61% (P<0.05) and additionally suppressed metastasis formation by 43%. In comparison to CXCR4 inhibition as a monotherapy, standard chemotherapy composed of cisplatin and etoposide reduced the growth of the primary tumor by 71% (P<0.01) but completely failed to suppress metastasis formation. Combination of chemotherapy and the CXCR4 inhibitor integrated the highest of both effects. The growth of the primary tumor was reduced to a similar extent as with chemotherapy alone and metastasis formation was reduced to a similar extent as with CXCR4 inhibitor alone. In conclusion, we demonstrate in this orthotopic mouse model that the addition of a CXCR4 inhibitor to chemotherapy significantly reduces metastasis formation. Thus, it might improve the overall therapy response and consequently the outcome of SCLC patients.

High-Resolution PET Imaging with Therapeutic Antibody-based PD-1/PD-L1 Checkpoint Tracers.

Checkpoint-blocking antibodies like those targeting the PD-1/PD-L1 pathway have revolutionized oncology. We developed radiotracers based on therapeutic checkpoint-blocking antibodies permitting sensitive and high-resolution PET imaging of both PD-1 and PD-L1 in immunocompetent mice. ImmunoPET of naive mice revealed similar overall expression patterns for PD-1 and PD-L1 in secondary lymphoid organs (spleen and lymph nodes). Interestingly, PD-L1 was also detected in brown adipose tissue (BAT), confirming the notion that BAT is immunologically relevant. Under pathophysiological conditions, strong expression of the receptor/ligand pair was also found in non-lymphoid tissues. Both were specifically detected in malignant tumors. PD-1 was readily detected after combined immunoradiotherapy causing massive tumor infiltration by PD-1+ lymphocytes. PD-L1 tracer uptake was reduced in PD-L1 knockout tumors. Moreover, monitoring the expression changes of PD-L1 in response to its main inducer, the effector T cell cytokine IFN-γ, revealed robust upregulation in the lung. This suggests that T cell responses in the lung, a vital organ continuously exposed to a variety of antigens, are strongly restrained by the PD-1 checkpoint. In turn, this could explain the association of PD-1 checkpoint inhibition with potentially fatal immune-mediated pneumonitis and partially also its efficacy in lung cancer.

An orthotopic mouse model of small cell lung cancer reflects the clinical course in patients.

Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with very poor prognosis due to early metastatic spread and development of chemoresistance. In the last 30 years the study of SCLC has been constrained by a lack of primary human tumor specimen thus highlighting the need of a suitable mouse model. In this article we present the establishment of an orthotopic xenograft mouse model which accurately reproduced the clinical course of SCLC. Orthotopic implantation enabled engraftment of primary lung tumors in all injected mice. Furthermore, immunodeficiency of mice allowed formation of spontaneous metastases in characteristic organs. Bioluminescence Imaging, Magnetic Resonance Imaging and Positron emission tomography were applied to monitor engraftment, metabolism and the exact growth of tumors over time. In order to mimic the extensive disease stage, mice were injected with aggressive human chemoresistant cells leading to development of chemoresistant tumors and early metastatic spread. As a proof of concept treatment of tumor-bearing mice with conventional chemotherapeutics reduced tumor volumes, but a complete regression of tumors was not achieved. By mimicking the extensive disease stage our mouse model can facilitate the study of mechanisms contributing to chemoresistance and metastasis formation, as well as drug screening and evaluation of new treatment strategies for SCLC patients.

Stereochemistry of amino acid spacers determines the pharmacokinetics of (111)In-DOTA-minigastrin analogues for targeting the CCK2/gastrin receptor.

The metabolic instability and high kidney retention of minigastrin (MG) analogues hamper their suitability for use in peptide-receptor radionuclide therapy of CCK2/gastrin receptor-expressing tumors. High kidney retention has been related to N-terminal glutamic acids and can be substantially reduced by coinjection of polyglutamic acids or gelofusine. The aim of the present study was to investigate the influence of the stereochemistry of the N-terminal amino acid spacer on the enzymatic stability and pharmacokinetics of (111)In-DOTA-(d-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-D) and (111)In-DOTA-(l-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-L). Using circular dichroism measurements, we demonstrate the important role of secondary structure on the pharmacokinetics of the two MG analogues. The higher in vitro serum stability together with the improved tumor-to-kidney ratio of the (d-Glu)6 congener indicates that this MG analogue might be a good candidate for further clinical study.

Effective Eradication of Glioblastoma Stem Cells by Local Application of an AC133/CD133-Specific T-cell-Engaging Antibody and CD8 T Cells.

Cancer stem cells (CSC) drive tumorigenesis and contribute to genotoxic therapy resistance, diffuse infiltrative invasion, and immunosuppression, which are key factors for the incurability of glioblastoma multiforme (GBM). The AC133 epitope of CD133 is an important CSC marker for GBM and other tumor entities. Here, we report the development and preclinical evaluation of a recombinant AC133×CD3 bispecific antibody (bsAb) that redirects human polyclonal T cells to AC133(+) GBM stem cells (GBM-SC), inducing their strong targeted lysis. This novel bsAb prevented the outgrowth of AC133-positive subcutaneous GBM xenografts. Moreover, upon intracerebral infusion along with the local application of human CD8(+) T cells, it exhibited potent activity in prophylactic and treatment models of orthotopic GBM-SC-derived invasive brain tumors. In contrast, normal hematopoietic stem cells, some of which are AC133-positive, were virtually unaffected at bsAb concentrations effective against GBM-SCs and retained their colony-forming abilities. In conclusion, our data demonstrate the high activity of this new bsAb against patient-derived AC133-positive GBM-SCs in models of local therapy of highly invasive GBM.

Does imaging αvß3 integrin expression with PET detect changes in angiogenesis during bevacizumab therapy?

UNLABELLED: In recent years, there has been a growing interest in molecular imaging markers of tumor-induced angiogenesis. Several radiolabeled RGD (arginine, glycine, aspartate) peptides have been developed for PET imaging of αvß3 integrins in the tumor vasculature, but there are only limited data on how angiogenesis inhibitors affect the tumor uptake of these peptides. METHODS: Changes in (68)Ga-NODAGA-c(RGDfK) peptide uptake were measured using PET during bevacizumab therapy of 2 αvß3-negative squamous cell carcinoma cell lines (A-431 and FaDu) that induce αvß3-positive neovasculature when transplanted into nude mice. Tumor uptake of (68)Ga-NODAGA-c(RGDfK) was correlated to microvascular density, vascular morphology, and permeability as well as αvß3 integrin expression. RESULTS: Bevacizumab significantly inhibited growth of A-431 tumors and caused a significant reduction in microvascular density and αvß3 integrin expression within 7 d after start of therapy. Bevacizumab also caused a normalization of blood vessel morphology and decreased tumor necrosis. However, (68)Ga-NODAGA-c(RGDfK) uptake was significantly increased at day 7 of therapy and did not decrease until after 3 wk of treatment. In Fadu xenografts, bevacizumab therapy caused only a minor inhibition of tumor growth and minor changes in (68)Ga-NODAGA-c(RGDfK) uptake. CONCLUSION: Uptake of radiolabeled RGD peptides is not necessarily decreased by effective antiangiogenic therapy. Early in the course of therapy a decrease in the expression of αvß3 integrins may not be reflected by a decrease in the uptake of RGD peptides.

Noninvasive positron emission tomography and fluorescence imaging of CD133+ tumor stem cells.

A technology that visualizes tumor stem cells with clinically relevant tracers could have a broad impact on cancer diagnosis and treatment. The AC133 epitope of CD133 currently is one of the best-characterized tumor stem cell markers for many intra- and extracranial tumor entities. Here we demonstrate the successful noninvasive detection of AC133(+) tumor stem cells by PET and near-infrared fluorescence molecular tomography in subcutaneous and orthotopic glioma xenografts using antibody-based tracers. Particularly, microPET with (64)Cu-NOTA-AC133 mAb yielded high-quality images with outstanding tumor-to-background contrast, clearly delineating subcutaneous tumor stem cell-derived xenografts from surrounding tissues. Intracerebral tumors as small as 2-3 mm also were clearly discernible, and the microPET images reflected the invasive growth pattern of orthotopic cancer stem cell-derived tumors with low density of AC133(+) cells. These data provide a basis for further preclinical and clinical use of the developed tracers for high-sensitivity and high-resolution monitoring of AC133(+) tumor stem cells.

Hybrid bombesin analogues: combining an agonist and an antagonist in defined distances for optimized tumor targeting.

Radiolabeled hybrid ligands with defined distances between an agonist and an antagonist for the gastrin-releasing peptide receptor were found to have excellent tumor-targeting properties. Oligoprolines served as rigid scaffolds that allowed for tailoring distances of 10, 20, and 30 Å between the recognition elements. In vitro and in vivo studies revealed that the hybrid ligand with a distance of 20 Å between the recognition elements exhibits the highest yet observed tumor cell uptake and retention time in prostate cancer cells.

Targeted radiotherapy of prostate cancer with a gastrin-releasing peptide receptor antagonist is effective as monotherapy and in combination with rapamycin.

UNLABELLED: The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer and is an attractive target for radionuclide therapy. In addition, inhibition of the protein kinase mammalian target of rapamycin (mTOR) has been shown to sensitize various cancer cells to the effects of radiotherapy. METHODS: To determine the effect of treatment with rapamycin and radiotherapy with a novel (177)Lu-labeled GRPr antagonist ((177)Lu-RM2, BAY 1017858) alone and in combination, in vitro and in vivo studies were performed using the human PC-3 prostate cancer cell line. PC-3 cell proliferation and (177)Lu-RM2 uptake after treatment with rapamycin were assessed in vitro. To determine the influence of rapamycin on (177)Lu-RM2 tumor uptake, in vivo small-animal PET studies with (68)Ga-RM2 were performed after treatment with rapamycin. To study the efficacy of (177)Lu-RM2 in vivo, mice with subcutaneous PC-3 tumors were treated with (177)Lu-RM2 alone or after pretreatment with rapamycin. RESULTS: Stable expression of GRPr was maintained after rapamycin treatment with doses up to 4 mg/kg in vivo. Monotherapy with (177)Lu-RM2 at higher doses (72 and 144 MBq) was effective in inducing complete tumor remission in 60% of treated mice. Treatment with 37 MBq of (177)Lu-RM2 and rapamycin in combination led to significantly longer survival than with either agent alone. No treatment-related toxicity was observed. CONCLUSION: Radiotherapy using a (177)Lu-labeled GRPr antagonist alone or in combination with rapamycin was efficacious in inhibiting in vivo tumor growth and may be a promising strategy for treatment of prostate cancer.

Inflammatory neovascularization during graft-versus-host disease is regulated by αv integrin and miR-100.

Acute graft-versus-host disease (GvHD) is a complex process involving endothelial damage and neovascularization. Better understanding of the pathophysiology of neovascularization during GvHD could help to target this process while leaving T-cell function intact. Under ischemic conditions, neovascularization is regulated by different micro RNAs (miRs), which potentially play a role in inflamed hypoxic GvHD target organs. We observed strong neovascularization in the murine inflamed intestinal tract (IT) during GvHD. Positron emission tomography imaging demonstrated abundant αvß3 integrin expression within intestinal neovascularization areas. To interfere with neovascularization, we targeted αv integrin-expressing endothelial cells, which blocked their accumulation in the IT and reduced GvHD severity independent of immune reconstitution and graft-versus-tumor effects. Additionally, enhanced neovascularization and αv integrin expression correlated with GvHD severity in humans. Expression analysis of miRs in the inflamed IT of mice developing GvHD identified miR-100 as significantly downregulated. Inactivation of miR-100 enhanced GvHD indicating a protective role for miR-100 via blocking inflammatory neovascularization. Our data from the mouse model and patients indicate that inflammatory neovascularization is a central event during intestinal GvHD that can be inhibited by targeting αv integrin. We identify negative regulation of GvHD-related neovascularization by miR-100, which indicates common pathomechanistic features of GvHD and ischemia.

Unexpected sensitivity of sst2 antagonists to N-terminal radiometal modifications.

Chelated somatostatin agonists have been shown to be sensitive to N-terminal radiometal modifications, with Ga-DOTA agonists having significantly higher binding affinity than their Lu-, In-, and Y-DOTA correlates. Recently, somatostatin antagonists have been successfully developed as alternative tracers to agonists. The aim of this study was to evaluate whether chelated somatostatin antagonists are also sensitive to radiometal modifications and how. We have synthesized 3 different somatostatin antagonists, DOTA-p-NO(2)-Phe-c[D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH(2), DOTA-Cpa-c[D-Cys-Aph(Hor)-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH(2) (DOTA-JR11), and DOTA-p-Cl-Phe-c[D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH(2), and added various radiometals including In(III), Y(III), Lu(III), Cu(II), and Ga(III). We also replaced DOTA with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) and added Ga(III). The binding affinity of somatostatin receptors 1 through 5 was evaluated in all cases. In all 3 resulting antagonists, the Ga-DOTA analogs were the lowest-affinity radioligands, with a somatostatin receptor 2 binding affinity up to 60 times lower than the respective Y-DOTA, Lu-DOTA, or In-DOTA compounds. Interestingly, however, substitution of DOTA by the NODAGA chelator was able to increase massively its binding affinity in contrast to the Ga-DOTA analog. The 3 NODAGA analogs are antagonists in functional tests. In vivo biodistribution studies comparing (68)Ga-DOTATATE agonist with (68)Ga-DOTA-JR11 and (68)Ga-NODAGA-JR11 showed not only that the JR11 antagonist radioligands were superior to the agonist ligands but also that (68)Ga-NODAGA-JR11 was the tracer of choice and preferable to (68)Ga-DOTA-JR11 in transplantable HEK293-hsst(2) tumors in mice. One may therefore generalize that somatostatin receptor 2 antagonists are sensitive to radiometal modifications and may preferably be coupled with a (68)Ga-NODAGA chelator-radiometal complex.

The influence of the combined treatment with Vadimezan (ASA404) and taxol on the growth of U251 glioblastoma xenografts.

BACKGROUND: One of the most important biological characteristics of Glioblastoma multiforme (GBM) is high vascular density. Vadimezan (ASA404, DMXAA) belongs to the class of small molecule vascular disrupting agents (VDA) that cause disruption of established tumor vessels and subsequent tumor hemorrhagic necrosis. Its selective antivascular effect is mediated by intratumoral induction of several cytokines including tumor necrosis factor-α (TNF-α), granulocyte-colony-stimulating factor (G-CSF), interleukin 6 (IL-6) and macrophage inflammatory protein 1α (MIP-1α). Preclinical studies have demonstrated that ASA404 acts synergistically with taxanes. In this study, we investigated if treatment of mice bearing U251 human glioblastoma xenografts with ASA404 and taxol may be synergistic. Therapy response was evaluated by measuring changes in tumor size and metabolic activity using 18F-FDG PET (Fluorodeoxyglucose - positron emision tomography) imaging. METHODS: U251 cells were inoculated s.c. in the right hind limb of NMRI-Foxn1nu athymic female nude mice. Animals were randomly assigned into 4 groups (7-9 animals/group) for treatment: control, taxol, ASA404, and ASA404 plus taxol. The animals received either a single dose of taxol (10 mg/kg), ASA404 (27.5 mg/kg), or taxol (10 mg/kg) plus ASA404 (27.5 mg/kg) administered i.p.; ASA404 was administred 24 h after the treatment with taxol. 4 and 24 h after treatment with ASA404 (28 and 48 h hours after treatment with taxol) 18 F-FDG PET scans were performed. RESULTS: The treatment with taxol did not affect the tumor growth in comparison to untreated controls. The treatment of animals with single dose ASA404 alone or in combination with taxol caused a significant delay in tumor growth. The combined treatment did not decrease the growth of the xenografts significantly more than ASA404 alone, but early changes in tumor 18 F-FDG uptake preceded subsequent growth inhibition. The tumor weights, which were determined at the end of treatment, were lower in case of combined treatment. CONCLUSIONS: The treatment with ASA404 alone or in combination with taxol showed antitumoral effects in our glioblastoma model probably through destruction of blood vessels. The implications for the anticancer effect of this compound warrant further preclinical studies. 18F-FDG PET appears to be a promising tool to monitor treatment with ASA404 early in the course of therapy.